Fluorescence examinations for the identification of specific substances have been known for a long time. The ability to emit light after photon absorption, i.e. to luminesce, is substance-specific. This is the basis of conventional luminescence analysis. Several million luminescent, i.e. fluorescing and/or phosphorescing organic compounds are known today, and it is often the case that several luminescent substances are present in a material that is to be examined. This often applies, for example, to measured samples and to issues encountered in biosciences and medicine. For example, human skin tissue contains at least ten different endogenous fluorophores, along with exogenous fluorophores, and consequently the autofluorescence spectrum of the skin is the result of many individual fluorescence bands. A number of methods are known, which generally have to be used in combination in order to yield a component analysis with fluorophore mixtures, for example, by varying the excitation wavelength, by turning to excitation spectra as a function of the fluorescence wavelengths, fluorescence decay behavior and polarization spectra, although employing combined methods is not only time-consuming but, for example, in cases where the fluorophore mixture is present in a matrix, might only be useable to a limited extent due to the optical properties of the matrix itself, such as self-absorption and scattering. Another complication of the analysis of fluorophore mixtures in matrices arises if the latter are non-homogeneous in terms of their optical properties and if the composition of the fluorophore mixture in these non-homogeneous matrices is additionally itself a function of the location. Such a situation exists in the matrix of human skin tissue, in view of the mixture of endogenous and exogenous fluorophores that is present there. The fluorophore component analysis with this matrix is also made more difficult in that it has a penetration depth for visible light that decreases sharply from the long-wave to the short-wave range. This drawback can be countered by non-linear fluorophore excitation by means of simultaneous two-photon absorption in the long-wave spectral range, but this considerably limits not only the above-mentioned broad combination of methods for the fluorophore component analysis and makes it extremely complicated, but above all, it also calls for the use of ultra-short, intense high-repeating laser light pulses in the femtosecond range (fs). This entails the well-known risk of photochemical bleaching of the fluorophores and, especially with in-vivo applications, there is also a risk of affecting the cell division rate caused by the requisite high photon flux densities of typically ≧1029 photons per cm2 and per second, and by the high-repeating radiation regime.
However, it is precisely the fluorophore component analysis in human tissue that is of considerable interest, e.g. in conjunction with medical-diagnostic, pharmaceutical and cosmetic issues. In particular, the focus of attention is directed at the endogenous fluorophore melanin. Melanin occurs, among other places, in the skin, hair and eyes; it is responsible, for example, for skin and hair color, and it especially plays a central role, on the one hand, as a “sunscreen” and, on the other hand, in the degeneration of skin tissue into malignant melanoma, the black skin cancer. According to S. P. Nighswander-Rempel et. al. in “A quantum yield map for synthetic eumelanin” in J. Chem. Phys. 123, 2005, 194901-1-6, when it comes to fluorescence analysis, melanin has the serious drawback of an extremely small fluorescence quantum yield in the order of magnitude of 10−4 at the maximum; even a specific fluorescence quantum yield derived from the unusual absorption of melanin is only in the order of magnitude of 10−6. The absorption spectrum of melanin differs from that of almost all other organic fluorophores. Whereas the latter exhibit only individual discrete absorption bands between the near ultraviolet and the near infrared spectral ranges, melanin exhibits a monotonously decreasing absorption curve in the cited spectral range. Thus, when two-photon absorption in the red or near infrared spectral range is applied to fluorophore mixtures containing melanin, the results do not even come close to achieving a selective excitation of the melanin spectrally because every light wavelength that excites any fluorophore also excites melanin. German patent specification DE 199 39 706 C2 discloses that an accumulation of the excited melanin in comparison to all other fluorophores can be achieved by two-photon excitation with femtosecond pulses, meaning that, so to speak, a certain compensation for the low fluorescence quantum yield is possible. This is based on the fact that two-photon excitation of melanin takes place as a stepwise process of two consecutive one-photon absorptions via a real intermediate level (see K. Teuchner et. al. in “Femtosecond Two-photon Excited Fluorescence of Melanin” in Photochem. Photobiol. 70(2), 1999, pp. 146-151), in contrast to the usual simultaneous two-photon excitation with an only virtual intermediate level in the case of the other relevant fluorophores. However, the fluorescence-spectroscopic significance and analytical usefulness of this accumulation of excited melanin are limited by its extremely low fluorescence quantum yield in comparison to the other relevant fluorophores. It is known from the publication by K. Hoffmann et. al. “Selective Femtosecond Pulse-Excitation of Melanin Fluorescence in Tissue” in J. Invest. Dermatol. 116 (2001), 629-630 that, with this two-photon excitation based on femtosecond pulses, a red shift of the fluorescence can be measured in malignant melanoma ex-vivo in comparison to healthy skin tissue and a shortening of the fluorescence decay occurs (also see German patent application DE 102 39 028 B4).
U.S. Pat. No. 5,034,613 describes a laser microscope with a simultaneous two-photon fluorescence excitation that, in order to examine cell material, uses excitation wavelengths in the range from red to near infrared, i.e. between 640 nm and 1200 nm, with pulse lengths in the sub-picosecond range, i.e. <10−12 seconds, here at 100 femtoseconds (fs), at a repetition rate of 80 MHz. A very high local light intensity arises due to the focusing at 1 μm. This very narrow focusing is meant to limit the bleaching of the fluorophores to the immediate observation area. Moreover, the two-photon excitation is supposed to suppress the so-called background fluorescence to a greater extent. German patent specification DE 44 14 940 C2 describes a luminescence scanning microscope using two-photon excitation that works with laser pulses that are greater than 1 picosecond (ps) in order to avoid the use of expensive femtosecond lasers. With an eye towards offsetting the low pulse power that is used so as to treat the examination objects gently, a greater measuring duration, i.e. a longer pulse sequence is used for the luminescence excitation. German patent application DE 197 19 344 A1 discloses an arrangement for the optical micromanipulation, analysis and processing of objects, said arrangement working with a wavelength spectrum for the excitation in the range between 400 nm and 1200 nm and pulse lengths in the nanosecond, picosecond and femtosecond ranges. The arrangement relates mainly to the use of a laser that can be tuned over the entire spectral range and less to the fluorescence excitation intended for the actual substance analysis. Nevertheless, this publication explicitly points out that only the pulse durations in the femtosecond range are used for the analysis. Pulse lengths in the range of picoseconds or longer are used exclusively for the micromanipulation.
German patent application DE 199 35 766 A1 describes a method for the optical excitation of fluorophore-marked DNA and RNA in which a simultaneous non-resonant multi-photon fluorescence excitation is used preferably at wavelengths in the range between 760 nm and 820 nm, and with power densities between 100 MW/cm2 und 10 TW/cm2. It is noted that the simultaneous two-photon or three-photon excitation is not known yet in the DNA/RNA analysis under discussion here. An example is presented in which various fluorophores with a wavelength of 770 nm, a pulse duration of 200 fs, a pulse frequency of 76 MHz and a power density of 500 GW/cm2 could be excited to a high-contrast fluorescence spectrum with maxima between 480 nm and 650 nm. German patent specification DE 199 39 706 C2 describes the selection of fluorophores for substance marking in multi-photon laser scanning microscopy, comprising a stepwise resonant absorption with real intermediate levels. Here, a much lower laser intensity, i.e. photon flux density, is said to be necessary for the excitation, so that, on the one hand, less equipment is needed and, on the other hand, the risk of electric disruptive discharges and the photochemical effect of bleaching of the substance sample can be minimized after the one-photon absorption. In particular, mention is made of synthetic melanin as such a fluorophore in which the mechanism of action of the stepwise resonant multi-photon absorption is systematically utilized, i.e. the excitation is not achieved via virtual but rather via real intermediate levels. Concretely speaking, a wavelength of 800 nm, a pulse duration of 120 fs and a pulse energy of 1 μJ are used for the fluorescence excitation. The emitted fluorescence is in the blue-green-red spectral range at a maximum of 610 nm. German patent application DE 100 65 146 A1 describes a method and an arrangement for non-invasive three-dimensional optical examination and treatment of the skin that, for the multi-photon excitation of the body's own fluorophores, use pulsed laser radiation in the near infrared range at wavelengths of 700 nm to 1200 nm as well as pulse lengths of less than 20 ps with light intensities in the order of magnitude between gigawatts per cm2 and terawatts per cm2 at a pulse sequence frequency of 80 MHz. In particular, it is said that melanoma of the skin can be located and irreversibly damaged. It is described that resonant and non-resonant multi-photon fluorescence excitation of specific endogenous fluorophores, especially melanin, occurs, as a result of which it is said to be possible to distinguish between certain pathological tissue and healthy tissue on the basis of the ascertained arrangement of the fluorescence intensity and of the fluorescence lifetime. The exact mechanisms of action of the multi-photon excitation in conjunction with the excitation parameters as well as the interpretation of the fluorescence response for purposes of precisely locating pathological tissue are not discussed.
International patent publication WO 02/069784 describes a portable fluorescence lifetime spectrometer (FLS) for the simultaneous in-vivo analysis of the spectral and temporal fluorescence properties of tissue or cells in terms of their carcinogenic or pre-carcinogenic tissue components. The time-dependent fluorescence response of endogenous fluorophores such as collagen, elastin, NADPH and tryptophan is highly dependent on the biochemical environment and on its pH value and oxygen content, as a result of which a conclusion can be drawn as to whether the tissue is said to be healthy or diseased. In less than one second, the FLS can process the data about the transient decay behavior of a certain frequency band of the fluorescence of the examined tissue over periods of time averaging 360 picoseconds and consequently, it is suitable for in-vivo use. This publication does not present a new measuring method but rather a measuring device that has been optimized for a specific purpose.
German patent application DE 102 39 028 B4 describes a method for identifying naturally occurring or synthetically produced types of melanin. The occurring melanin is selectively excited—relative to other fluorophores present in the sample—by one-photon excitation and by stepwise, resonant two-photon excitation with laser pulses having a wavelength of 800 nm and at a pulse length in the femtosecond range, and the fluorescence spectrum obtained as the response to this is evaluated after spectral distribution and after being temporally resolved. On the basis of the spectral distribution of the obtained fluorescence intensities and of the decay behavior, it becomes possible to selectively distinguish among the various types of melanin and thus to draw a conclusion about the presence of tissue that is suspected of having a malignant melanoma.
In the state of the art, fluorophores in general and melanin in particular are regularly detected with laser pulses having pulse lengths in the femtosecond range, but at the most of less than 20 ps. The wavelength range is specified as being from 700 nm to 1200 nm, a wavelength of 800 nm being commonly used. The high-energy pulses are radiated highly repetitively at frequencies of, for example, 80 MHz, and they generate photon flux power densities that lie between 100 GW/cm2 and several TW/cm2.